Cell populations which co-express CD49c and CD90

ABSTRACT

Substantially homogenous cells populations which co-express CD49c, CD90 and telomerase are made. In one embodiment, humans suffering from a degenerative, traumatic, acute injury, cardiac or neurological condition are treated with the substantially homogenous cells populations which co-express CD49c, CD90 and telomerase. In another embodiment, committed progenitor cells are made are made by selecting from a cultured source of a cell population which co-express CD49c and CD90 and modifying the cell population. The committed progenitor cells can be employed to treat a human suffering from a degenerative, traumatic, acute injury, cardiac or neurological condition and formulate pharmaceutical compositions.

RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.09/960,244, filed Sep. 21, 2001. The entire teachings of the aboveapplication are incorporated herein by reference.

BACKGROUND OF THE INVENTION

A number of conditions and diseases of the central (brain and spinalcord) and peripheral nervous system adversely affect humans. Theseconditions and diseases include, for example, spinal cord injury,amyotrophic lateral sclerosis (ALS), Parkinson's disease, stroke,traumatic brain injury, brain tumors and Fabry Disease. Clinicalmanagement strategies frequently focus of the prevention of furtherneurological damage or injury rather than replacement or repair of thedamaged neurological tissue (e.g., neurons, glial cells); includetreatment with exogenous steroids and synthetic, non-cellularpharmaceutical drugs; and have varying degrees of success which maydepend on the continued administration of the steroid or synthetic drug.

For example, the majority of spinal cord injuries are compressioninjuries with the remaining cases involving complete transection of thespinal cord. Current therapeutic treatments for spinal cord injuryinclude the prevention of additional spinal cord injury by physicallystabilizing the spine through surgical and non-surgical procedures andby inhibiting the inflammatory response with steroidal therapy. Thus,there is a need to develop new, improved and effective methods oftreatment for neurological diseases and conditions in humans.

SUMMARY OF THE INVENTION

The present invention relates to a population of cells which co-expressCD49c and CD90 and methods of treating neurological conditions in humanswith these populations of cells.

In one embodiment, the invention is a substantially homogenous cellpopulation which co-express CD49c, CD90 and telomerase.

In another embodiment, the invention is a substantially homogenous cellpopulation which co-express CD49c and CD90, but does not express bonesialoprotein (BSP).

In still another embodiment, the invention includes a method of making asubstantially homogenous cell population which co-express CD49c and CD90by culturing a source of the cell population a seeding cell density ofless than about 100 cells/cm² under a low oxidative stress condition andselecting from the cultured source of the cell population, cells whichco-express CD49c and CD90.

In a further embodiment, the invention includes a method of making asubstantially homogenous cell population which co-express CD49c and CD90by culturing a source of the cell population at a seeding density ofless than about 100 cells/cm² under a low oxidative stress condition;and selecting from the cultured source of the cell population, cellswhich co-express CD49c and CD90.

In another embodiment, the invention is a method of making asubstantially homogenous cell population which co-express CD49c and CD90by culturing a source of the cell population at a seeding density ofless than about 50 cells/cm² under a low oxidative stress condition; andselecting from the cultured source of the cell population, cells whichco-express CD49c and CD90.

In still another embodiment, the invention is a method of making asubstantially homogenous cell population which co-express CD49c and CD90by culturing a source of the cell population at a seeding density ofless than about 30 cells/cm² under a low oxidative stress condition; andselecting from the cultured source of the cell population, cells whichco-express CD49c and CD90.

A further embodiment of the invention is a method of making asubstantially homogenous cell population which co-express CD49c and CD90by culturing a source of the cell population at a seeding density ofless than about 75,000 cells/cm² under a low oxidative stress conditionto produce an adherent cell population and culturing the adherent cellpopulation at a seeding density of less than about 100 cells/cm² under alow oxidative stress condition. Cells which co-express CD49c and CD90are selected from the cultured adherent cell population.

Another embodiment of the invention includes a method of making asubstantially homogenous cell population which co-express CD49c and CD90by culturing a source of the cell population at a seeding cell densityof less than about 50 cells/cm² under a low oxygen condition andselecting from the cultured source of the cell population, cells whichco-express CD49c and CD90.

In yet another embodiment, the invention includes a method of making asubstantially homogenous cell population which co-express CD49c and CD90by culturing a source of the cell population at a seeding cell densityof less than about 30 cells/cm² under a low oxygen condition andselecting from the cultured source of the cell population, cells whichco-express CD49c and CD90.

In a further embodiment, the invention is a method of making asubstantially homogenous cell population which co-express CD49c and CD90by culturing a source of the cell population at a seeding cell densityof less than about 75,000 cells/cm² under a low oxygen condition toproduce an adherent cell population; culturing the adherent cellpopulation at an initial density of less than about 100 cells/cm² undera low oxygen condition; and selecting from the cultured adherent cellpopulation, cells which co-express CD49c and CD90.

Another embodiment of the invention is a method of treating a humansuffering from a degenerative or acute injury condition, comprising thestep of administering to the human a substantially homogenous cellpopulation which co-express CD49c and CD90.

In yet another embodiment, the invention includes a method of treating ahuman suffering from a neurological condition, comprising the step ofadministering to the human a substantially homogenous cell populationwhich co-express CD49c and CD90.

In still another embodiment, the invention is a method of treating ahuman suffering from a cardiac condition to the human a substantiallyhomogenous cell population which co-express CD49c and CD90.

An additional embodiment of the invention is a method of treating ahuman suffering from a neurological condition by culturing a source of acell population at a seeding cell density of less than about 100cells/cm² under a low oxygen condition; selecting from the culturedsource of the cell population, a population of cells which co-expressCD49c and CD90; and administering the population of cells whichco-express CD49c and CD90 to the human.

In yet an additional embodiment, the invention includes a method oftreating a human suffering from a neurological condition, comprisingculturing a source of a cell population; selecting from the culturedsource of the cell population, a population of cells which co-expressCD49c and CD90; and administering the population of cells whichco-express CD49c and CD90 to the human.

In still another embodiment, the invention is a method of making acommitted progenitor cell, comprising culturing a source of a cellpopulation; selecting from the cultured source of the cell population,cells which co-express CD49c and CD90; and modifying the cells whichco-express CD49c and CD90 to become committed progenitor cells.

An additional embodiment of the invention includes a method of treatinga human suffering from a neurological condition, comprising culturing asource of a cell population; selecting from the cultured source of thecell population, cells which co-express CD49c and CD90; modifying thecells which co-express CD49c and CD90 to become a committed progenitorcell; and administering the modified cells to the human.

In another embodiment, the invention relates to a method of treating ahuman suffering from a degenerative or acute injury condition byadministering to the human a substantially homogenous cell populationwhich co-express CD49c, CD90 and telomerase.

In another embodiment, the invention provides a pharmaceuticalcomposition comprising a substantially homogenous cell population whichco-express CD49c and CD90.

In yet another embodiment, the invention provides a a pharmaceuticalcomposition comprising a substantially homogenous cell population whichco-express CD49c, CD90 and telomerase.

A further embodiment of the invention is a method of treating a humansuffering from a neurological condition, comprising the step ofadministering to the human a substantially homogenous cell populationwhich co-express CD49c, CD90 and telomerase.

In another embodiment, the invention is a method of treating a humansuffering from a degenerative or acute injury condition, comprising thestep of administering to the human a substantially homogenous cellpopulation which co-express CD49c and CD90, but does not express bonesialoprotein (BSP).

In yet another embodiment, the invention is a method of treating a humansuffering from a neurological condition, comprising the step ofadministering to the human a substantially homogenous cell populationwhich co-express CD49c and CD90, but does not express bone sialoprotein.

The invention described herein provides a substantially homogenouspopulation of cells for treating a condition or disease in a human.Advantages of the cell based therapies of the claimed invention include,for example, incorporation of the cells into the tissue (e.g., centralnervous system tissue, peripheral nervous system tissue, cardiactissue); the incorporated cells have the potential to differentiate ordevelop into neuronal, glial or other cells (e.g., cardiac muscle) toreplace or facilitate repair of the damaged, traumatized or degeneratingtissue thereby resulting in a more permanent treatment of thedegenerative, acute injury, traumatized, neurological or cardiaccondition; and the ability to employ characterized reproduciblepopulations of cells in treatment regimens. The cells of the inventionhave the potential to secret beneficial cytokines and trophic factors(e.g., BDNF, IL-6, NGF and MCP-1).

Thus, treatment of humans with populations of cells which co-expressCD49c and CD90 can potentially reverse, diminish or repair the loss dueto a degenerative, acute injury, neurological or cardiac condition in ahuman, thereby increasing the quality of life and life expectancy forthe human.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B and 1C illustrate the flow cytometric analysis of cellpopulations in the Master and Working Cell Banks generated from a bonemarrow aspirate following red blood cell lysis.

FIGS. 2A, 2B, and 2C illustrate the flow cytometric analysis of cellpopulations in the Master and Working Cell Banks generated from a bonemarrow aspirate following density separation.

FIG. 3 illustrates the yield (Cell Number) of cells that co-expressCD49c and CD90 cells during ex vivo expansion of a Primary Cell Bank ofcolony forming units (CFUs) derived from human bone marrow aspirates.

FIG. 4 illustrates the doubling rate of cell populations that co-expressCD49c and CD90 cell population in culture.

FIG. 5 illustrates the Basso-Beattie-Bresnahan (BBB) index for ratsfollowing spinal cord injury and after transplantation with asubstantially homogenous cell population which co-express CD49c andCD90. At 19 days post contusion, rat received the cell transplantationshowed greater improvement than rat received only PBS control.

DETAILED DESCRIPTION OF THE INVENTION

The features and other details of the invention, either as steps of theinvention or as combinations of parts of the invention, will now be moreparticularly described and pointed out in the claims. It will beunderstood that the particular embodiments of the invention are shown byway of illustration and not as limitations of the invention. Theprinciple features of this invention can be employed in variousembodiments without departing from the scope of the invention.

The present invention relates to a substantially homogeneous cellpopulation of cells which co-express CD49c and CD90. The invention alsorelates to a substantially homogeneous cell population of cells whichco-express CD49c, CD90 and telomerase. The invention further relates toa substantially homogeneous cell population of cells which co-expressCD49c and CD90, but does not express bone sialoprotein (BSP).

“Substantially homogenous” as used herein refers to a population ofcells wherein the majority (e.g., between about 100% to about 70%) ofthe total number of cells have a specified characteristic of interest(e.g., co-express CD49c and CD90; co-express CD49c, CD90 and telomerase;co-express of CD49c and C90 with minimal expression of CD34 and/orCD45).

In one embodiment, the substantially homogenous population of cellswhich co-express CD49c and CD90 is a population of cells wherein betweenabout 80% to about 90% of the cells co-express the cell surface antigensCD49c and CD90. In another embodiment, the substantially homogenouspopulation of cells is a population of cells wherein between about 70%to about 80% of the cells co-express the cell surface antigens CD49c andCD90.

“Co-express,” as used herein, refers to the simultaneous detection oftwo or more molecules, e.g., CD49c and CD90, on or in a single cell.Techniques to detect co-expression of CD49c and CD90 in cells (e.g.,bone marrow stromal cells) are well established. For example,co-expression of CD49c and CD90 on a cell can be detected by multiplecolor cytometric analysis. CD49c can be detected employing a fluoresceinlabeled probe and CD90 can be detected employing a Texas red probe. TheCD49c and CD90 cell surface antigens can be visualized with the aid of aflow cytometer equipped with multiple filters capable of detecting themultiple colors. Techniques to detect the molecules of interest can alsoinclude ELISA, RIA, immunoflouresence microscopy and quantitative PCR.

In another embodiment, the invention is a substantially homogenous cellpopulation of the invention which co-express CD49c and CD90, but doesnot express bone sialoprotein.

The substantially homogenous cell population of the invention whichco-express CD49c and CD90 has a doubling time less than between about144 hours to about 48 hours. In one embodiment, the doubling time of thecell population is less than about 144 hours. In another embodiment, thedoubling time of the cell population is less than about 72 hours. Instill another embodiment, the doubling time is less than about 48 hours.The doubling time of the cells of the invention can be varied dependingon, for example, the density of the cells in culture (e.g., 100cells/cm²) and/or the concentration of oxygen employed to culture thecells (e.g., a low oxygen concentration such as about 5% oxygen).

The substantially homogenous cell population which co-express CD49c andCD90 can have the potential to differentiate into a preselectedphenotype (e.g., chondrocytes, astrocytes, oligodendrocytes, neurons,bone, osteoclasts, osteoblasts, cardiomyocytes, pancreatic islet cells,skeletal muscle, smooth muscle, hepatocytes and retinal ganglial cells).The potential to differentiate into a preselected phenotype refers tothe ability of the cell population to change to a functional cell type.

The substantially homogenous cell population which co-express CD49c andCD90 do not, after between about 20 population doublings to about 50population doublings, substantially express at least one cell senescentmarker selected from a group consisting of P21 and P53. A senescentmarker would be any marker associated with senescence or aging in a cell(e.g., P21, P53). The senescent marker can be a cytoplasmic, nuclear orcell surface marker.

In one embodiment, the cells of the invention undergo about 20population doublings and still co-express CD49c and CD90 but do notsubstantially express at least one cell senescent marker selected from agroup consisting of P21 and P53. In another embodiment, the cells of theinvention undergo about 30 population doubles and still co-express CD49cand CD90 but do not substantially express at least one cell senescentmarker selected from a group consisting of P21 and P53. In yet anotherembodiment, the cells of the invention undergo about 40 populationdoublings and still co-express CD49c and CD90 but do not substantiallyexpress at least one cell senescent marker selected from a groupconsisting of P21 and P53. In still another embodiment, the cells of theinvention undergo about 50 population doublings and still co-expressCD49c and CD90 but do not substantially express at least one cellsenescent marker selected from a group consisting of P21 and P53. One ofskill in the art would be able to determine when a cell has undergone apopulation doubling (Freshney, R. I. “Culture of Animal Cells: A Manualof Basic Techniques” New York, Wiley-Liss (1994)) and be able todetermine whether the cell populations co-express CD49c and CD90 and donot substantially express at least one cell senescent marker selectedfrom a group consisting of P21 and P53 employing established techniques(e.g., flow cytometry, quantitative PCR).

The substantially homogenous cell population of the invention whichco-express CD49c and CD90 can further include expression of P21 or P53after between about 20 to about 50 population doublings of the cells.Expression of a senescent marker (e.g., P21, P53) is a relativeexpression of the senescent marker (e.g., relative to 18s rRNA GAPDH(Glyceraldehyde-3-phosphate dehydrogenase), actin). “Relativeexpression,” as used herein, is expression (e.g., nucleic acid, protein)of a molecule of interest (e.g., CD49c, CD90, telomerase, CBFA1, BSP,BDNF, IL-6, MCP-1) with respect to expression of a standard or referencemarker (e.g. 18s rRNA, actin, GFAP). In a preferred embodiment,expression of P53 is a relative expression of up to about 3000transcripts of P53 (e.g., 0, 100, 1000, 1500, 2000) per 10⁶ transcriptsof an 18s rRNA and expression of P21 is a relative expression of up toabout 20,000 transcripts of P21 per 10⁶ transcripts of an 18s rRNA.

In one embodiment, the expression of p53 is about 3000 transcripts ofp53 per 10⁶ transcripts of an 18s rRNA. In another embodiment, theexpression of p53 is about 2000 transcripts of p53 per 10⁶ transcriptsof an 18s rRNA. In yet another embodiment, the expression of p53 isabout 1000 transcripts of p53 per 10⁶ transcripts of an 18s rRNA.

In another embodiment, the expression of p21 is up to about 20,000transcripts of p21 (e.g., 0, 100, 1000, 5000, 10000, 15000, 20000) per10⁶ transcripts of an 18s rRNA. In still another embodiment, theexpression of p21 is about 15,000 transcripts of p21 per 10⁶ transcriptsof an 18s rRNA. In yet another embodiment, the expression of p21 isabout 500 transcripts of p21 per 10⁶ transcripts of an 18s rRNA.

In one embodiment, the expression of a bone lineage marker core bindingfactor 1 (CBFA1) (Otto, F. et al., Cell 89(5) 765-771 (1997)) is about5000 transcripts of the bone lineage marker per 10⁶ transcripts of an18s rRNA. In another embodiment, the expression of the bone lineagemarker CBFA1 is about 3000 transcripts of the bone lineage marker per10⁶ transcripts of an 18s rRNA. In still another embodiment, theexpression of the bone lineage marker CBFA1 is about 1000 transcripts ofthe bone lineage marker per 10⁶ transcripts of an 18s rRNA.

The substantially homogenous cell population of the invention can be acell population from any human tissue (e.g., bone marrow, fat, skin,placenta, muscle, umbilical cord blood). In a preferred embodiment, thesubstantially homogeneous cell population is derived from bone marrowcells (e.g., human bone marrow stromal cells). Cells of the inventioncan be referred to as “derived” from any human tissue. Cells derivedfrom tissues can be obtained, for example, by lysis of the source of thecells (e.g., bone marrow cells). For example, bone marrow stromal cellsare derived from whole bone marrow aspirates after ammonium chloridelysis of the bone marrow aspirates. The ammonium chloride removes redblood cells from the aspirates and the resulting cell pellet is employedto generate the substantially homogenous cell population whichco-express CD49c and CD90 cells of the invention. Alternatively, thebone marrow can be processed (e.g., fractionated by density gradientcentrifugation, NH₂Cl lysis, fluorescent activated sorting or magneticsorting) to derive the cell populations of the invention. For example,the bone marrow aspirates or lysed bone marrow cells are passed througha density gradient to separate the cells of the invention from cellulardebris as a result of lysis. Alternatively, or additionally, the bonemarrow aspirates or lysed bone marrow cells can form a density gradient.

Whole bone marrow aspirates are obtained from a human and cultured incontact with a solid phase. Alternatively, or additionally, the wholebone marrow aspirate can be processed to yield a mononuclear cellfraction which is the cultured in contact with a solid phase. The solidphase can be plastic (e.g., tissue culture treated plastics).

The mononuclear cell fraction can be obtained from a whole bone marrowaspirate on a density gradient by established procedures. Alternatively,the mononuclear cell fraction is obtained by lysis of the red bloodcells contained in the bone marrow aspirate. The lysis is done by mixingthe bone marrow aspirate with ammonium chloride.

Human bone marrow cells are obtained from healthy human donors byaspirations of the iliac crest and bone marrow stromal cell populationsobtained employing well established techniques. For example,substantially homogenous cell populations which co-express CD49c andCD90 are obtained from human iliac crest bone marrow aspirates andprocessed to mononuclear cell fractions from which bone marrow stromalcells are selectively propagated in vitro based upon their propensity toattach to plastic and divide in response to defined cell culture medium.The plastic-adherent cells are optimally grown at a cell concentrationthat encourages virtually only the self-renewing cells, referred to ascolony-forming unit fibroblast-like cells (Cfu-f), to proliferate. TheCfu-f-derived cells are analyzed for cells which co-express CD49c andCD90 and sub-cultured to produce a substantially homogenous cellpopulation which co-express CD49c and CD90.

The bone marrow aspirate, or a cellular fraction of the bone marrowaspirate, is cultured in contact with a solid phase and an intermediatecell population is isolated from the resulting cell culture based ontheir propensity to adhere to the solid phase. Bone marrow aspirates, ora cellular fraction of the aspirate, are cultured at a dissolved oxygenconcentration of less than about 20%, preferably between about 1% toabout 10%, and most preferably from between about 2% oxygen to about 7%oxygen. In a preferred embodiment, the dissolved oxygen concentration isabout 5% oxygen. The resulting adherent cell population is expanded toyield a substantially homogeneous cell population which co-express CD49cand CD90.

Bone marrow cell expansion is conducted with a seeding density of lessthan about 2500 cell/cm², preferably less than about 1000 cells/cm², andmost preferably less than about 100 cells/cm². In a particularembodiment, the initial cell density in the expansion step is betweenabout 30 cells/cm² to about 50 cells/cm². A seeding density would be thenumber of adherent cells per cm² obtained from mononuclear bone marrowcells.

Standard media preparations can be used to culture the bone marrowcells. For example, the media can be minimum essential medium-alphamodification supplemented with 4 mM L-glutamine and 0 to 10% lotselected fetal bovine serum (FSB), preferably about 10% FSB. Theculturing step can be conducted for any reasonable period, for example,between about 3 to about 25 days and most preferably between about 3 toabout 15 days.

An intermediate cell population is isolated from the cell culturedescribe above based on its propensity to adhere to the solid phase. Theintermediate cell population is grown at a cell concentration thatencourages virtually only the self-renewing cells, referred to herein ascolony-forming unit fibroblast-like cells (Cfu-f), to proliferate. TheCfu-f-derived cells are sub-cultured under defined conditions to producea substantially homogeneous population of cells (Example 1). Accordingto the invention, the expansion yields a substantially homogeneous cellpopulation which co-express CD 49 and CD 90.

In another embodiment, the substantially homogenous cell population doesnot express CD34 and/or CD45. The presence or absence of CD34 and CD45can be detected on bone marrow mononuclear cells which co-express CD49cand CD90 using routine methods including, for example, antigen-specificELISA assays, quantitative PCR, or flow cytometry. Cells whichco-express CD49c and CD90, but do not express either or both CD34 and/orCD45, are propagated in culture and stored until use in the methods ofthe invention.

In yet another embodiment, the substantially homogenous population ofcells co-expressing CD49c and CD90 express a trophic factor selectedfrom the group consisting of brain-derived neurotrophic factor (BDNF)(Barde, Y. A., et al. EMBO J., 1(5):549-553 (1982)), nerve growth factor(NGF) (Levi-Montalcini, R., Arch Biol 76(2):387-417 (1965)),neurotrophin-3 (NT-3) (Mohn, A., et al., Nature 344:339-341 (1990)),interleukin-6 (IL-6) (Barton, B. E., Clin. Immumol. Immunopathol.85(1):16-20 (1997)), interleukin-7 (IL-7), interleukin-11 (IL-11), stemcell factor (SCF), macrophage chemoattractin protein-1 (MCP-1), matrixmetalloproteinase-9 (MMP-9) and Cystatin-C.

Expression of BDNF, NGF, NT-3, IL-6, IL-7, IL-11, SCF, MCP-1, MMP-9 andCystatin-C in substantially homogenous populations of cellsco-expressing CD49c and CD90 can be augmented by a variety oftechniques, including ex vivo cultivation of the cells in chemicallydefined medium.

In yet another embodiment, the invention is a substantially homogenouscell population which co-express CD49c, CD90 and telomerase. Expressionof telomerase is a relative expression, for example, a relativeexpression of greater than between about 1 transcript of telomerase per10⁶ transcripts of an 18s rRNA and about 10 transcripts of telomeraseper 10⁶ transcripts of an 18s rRNA. In one embodiment, expression oftelomerase is about 1 transcript of telomerase per 10⁶ transcripts of an18s rRNA. In another embodiment, expression of telomerase is about 5transcripts of telomerase per 10⁶ transcripts of an 18s rRNA. In yetanother embodiment, expression of telomerase is about 10 transcripts oftelomerase per 10⁶ transcripts of an 18s rRNA.

The cell population which co-express CD49c, CD90 and telomerase has adoubling time of less than about 144 hours, less than about 72 hours orless than about 48 hours.

The cell population which co-express CD49c, CD90 and telomerase has thepotential to differentiate into a preselected phenotypes (e.g., achondrocyte, an astrocyte, an oligodendrocyte, a neuron, osteocyte,osteoblast, osteoclast, a cardiomyocyte, a pancreatic islet cell, askeletal muscle, a smooth muscle, a hepatocyte and a retinal ganglialcell).

The cell population which co-express CD49c, CD90 and telomerase canfurther include expression of P21 or P53 after between about 20 to about50 population doublings of the cells (e.g., 20, 30, 40 or 50 populationdoublings). Expression of P53 is a relative expression of up to about3000 transcripts of P53 per 10⁶ transcripts of an 18s rRNA (e.g, 3000,2000 or 1000 transcripts of P53 per 10⁶ transcripts of an 18s rRNA).Expression of P21 is a relative expression of up to about 20,000transcripts of P21 per 10⁶ transcripts of an 18s rRNA (e.g., 20000,15000 or 5000 transcripts of P21 per 10⁶ transcripts of an 18s rRNA).

In another embodiment, the cell population which co-express CD49c, CD90and telomerase does not express CD34 and/or CD45. In yet anotherembodiment, the cell population which co-express CD49c, CD90 andtelomerase express at least one trophic factor selected from the groupconsisting of BDNF, IL-6 and MCP-1.

In still another embodiment, the invention includes a method of making asubstantially homogenous cell population which co-express CD49c and CD90comprising culturing a source of the cell population (e.g., human bonemarrow cells) and selecting from the cultured source of the cellpopulation, cells which co-express CD49c and CD90. In one embodiment,the source of the cell population is cultured under a low oxygencondition (e.g., less than atmospheric). “Low oxygen condition,” as usedherein, refers to a concentration (e.g., percent of oxygen based onvolume, weight or molarity) which is less than atmospheric oxygen.

In still another embodiment, the invention includes a method of making asubstantially homogenous cell population which co-express CD49c, CD90and telomerase comprising culturing a source of the cell population(e.g., human bone marrow cells) and selecting from the cultured sourceof the cell population, cells which co-express CD49c, CD90 andtelomerase.

In a further embodiment, the invention includes a method of making asubstantially homogenous cell population which co-express CD49c and CD90but does not express bone salioprotein comprising culturing a source ofthe cell population and selecting from the cultured source of the cellpopulation, cells which co-express CD49c, CD90 and a bone lineagemarker.

In a preferred embodiment, the low oxygen condition is an oxygenconcentration less than about 15% oxygen, and more preferably an oxygenconcentration less than about 10%, and most preferably an oxygenconcentration of about 5% oxygen. In another embodiment, the source ofthe cell population is cultured at a seeding density of less than about100 cells/cm² (e.g., 95, 90, 80, 50, 30, 25 cells/cm²) under low oxygenconditions (e.g., less than atmospheric, 5% oxygen).

In an additional embodiment, the invention includes a method of making asubstantially homogenous cell population which co-express CD49c and CD90comprising culturing a source of the cell population (e.g., human bonemarrow cells) and selecting from the cultured source of the cellpopulation, cells which co-express CD40c and CD90 by culturing thesource of the cell population under low oxidative stress (e.g.,glutathione, Vitamin C, Catalase, Vitamin E, N-Acetylacysteine). “Lowoxidative stress,” as used herein, refers to conditions of no or minimalfree radical damage to the cultured cells.

The method of making a substantially homogenous population of cellswhich co-express CD49c and CD90; co-express CD49c, CD90 and telomerase;or co-express CD49c, CD90 but does not express bone salioprotein (BSP),can further include lysing the source of the cell population (e.g., bonemarrow aspirates) prior to culturing the source of the cell population.For example, lysis of a bone marrow aspirate can result in the lysis ofhematopoietic cells leaving the non-hematopoietic cells un-lysed.Additionally, or alternatively, the method can further includefractionating (e.g., by passage through or formation of a densitygradient, by NH₂Cl lysis) the source of the cell population (e.g., bonemarrow aspirates) prior to culturing the source of the cell population.

The cells made by the method of the invention can also express at leastone trophic factor (e.g., BDNF, NGF, NT-3, IL-6, IL-7, IL-11, SCF,MCP-1, MMP-9 and Cystatin-C). In another embodiment, the substantiallyhomogenous population of cells which co-express CD49c and CD90;co-express CD49c, CD90 and teleromerase; co-express CD49c, CD90 but doesnot express bone salioprotein, made by the method of the invention donot express CD34 and/or CD45.

In still another embodiment, the invention is a method of treating ahuman suffering from a degenerative or acute injury condition,comprising the step of administering to the human a substantiallyhomogenous cell population which co-express CD49c and CD90. The cellsused to treat the human suffering from a degenerative or acute injurycondition can also not express CD34 and/or CD45.

Degenerative disease is a disease in which the decline (e.g., function,structure, biochemistry) of particular cell type (e.g., neuronal,muscle, connective, epithelial) results in an adverse clinicalcondition. For example, Parkinson's disease is a degenerative disease inthe central nervous system (e.g., basal ganglia) which is characterizedby rhythmical muscular tremors, rigidity of movement, festination,droopy posture and masklike facies. Degenerative diseases that can betreated with the substantially homogenous cell populations of theinvention which co-express CD49c and CD90 can be, for example,Parkinson's disease, Huntington's disease, Alzheimer's disease,amyotrophic lateral sclerosis, congenital heart failure, cardiomyopathy,ataxias, and spinal muscular dystrophy.

An acute injury condition is a condition in which an event or multipleevents results in an adverse clinical condition. The event which resultsin the acute injury condition can be an external event such as bluntforce or compression or an internal event such as sudden ischemia (e.g.,stroke or heart attack). Acute injury conditions that can be treatedwith the substantially homogenous cell populations of the inventionwhich co-express CD49c and CD90; which co-express CD49c, CD90 andtelomerase; which co-express CD49c and CD90, but does not express bonesialoprotein (BSP), can be, for example, spinal cord injury, traumaticbrain injury, myocardial infarction and stroke.

In a further embodiment, the invention includes a method of treating ahuman suffering from a cardiac condition, comprising the step ofadministering to the human a substantially homogenous cell populationwhich co-express CD49c and CD90. A cardiac condition is a disease of theheart. The disease of the heart can be a disease of the cardiac muscle,connective tissue of vessels of the heart. The cells used to treat thehuman suffering from a cardiac condition can also not express CD34and/or CD45. A cardiac condition that can be treated by the cells of theinvention can be, for example, myocardial infarction, myocarditis,vascular heart disease, cardiomyopathy, congenital heart disease,eschemic heart disease, heart transplant and pre-transplantation bridge.

An additional embodiment of the invention includes a method of treatinga human suffering from a neurological condition, comprising the step ofadministering to the human a substantially homogenous cell populationwhich co-express CD49c and CD90; co-express CD49c, CD90 and telomerase;CD49c, CD90 and a bone lineage marker. “A neurological condition,” asused herein, refers to any state of the nervous system (central orperipheral nervous system) which deviates in any manner from a normalnervous system or nervous system of a mammal (e.g., human) not affectedby a neurological condition. The neurological condition can be acondition of the central (brain or spinal cord) or peripheral nervoussystem. The neurological condition can be, for example, the result orconsequence of a disease (e.g., amyotrophic lateral sclerosis,Parkinson's Disease, Fabry Disease), acute injury condition (e.g.,stroke, brain injury, spinal cord injury) or a combination of diseaseand acute injury condition. Other neurological conditions which can betreated with the substantially homogenous population of cells of theinvention which co-express CD49c and CD90 include, for example,metachromatic distropy, adrenal leukodystrophy, Canavan disease,Pelizaeus Merzbacher, Nieman-pick and a brain tumor.

In still another embodiment, the invention includes a method of treatinga human suffering from a neurological condition, comprising culturing(e.g., low oxygen conditions; oxygen conditions less than atmospheric;about 5% oxygen) a source of a cell population (e.g., bone marrow, fat,cord blood, skin) and selecting from the cultured source of the cellpopulation, a population of cells which co-express CD49c and CD90. Theselected population of cells which co-express CD49c and CD90 areadministered to the human.

In one embodiment, the substantially homogenous population of cellswhich are administered to the human co-express CD49c and CD90 and lackCD34 and/or CD45. In another embodiment, the substantially homogenouspopulation of cells which are administered to the human co-expressCD49c, CD90 and telomerase. In yet another embodiment, the substantiallyhomogenous population of cells which are administered to the humanco-express CD49c and CD90 or co-express CD49c, CD90 and telomerase andexpress at least three trophic factors selected from the groupconsisting of BDNF, NGF, NT-3, IL-6, IL-7, IL-11, SCF, MCP-1, MMP-9 andCystatin-C (e.g., BDNF, IL-6 and MCP-1).

The synthesis and secretion of cytokines and trophic factors from thesubstantially homogenous population of cells of the invention canprotect surrounding cells near or distant from the site oftransplantation from further damage as a consequence of thedegenerative, acute injury or neurological condition. The synthesis andsecretion of cytokines and trophic factors from the substantiallyhomogenous population of cells of the invention can also, oralternatively, promote regeneration of cells and tissues of the host(e.g., human suffering from a acute injury, neurological, cardiac ordegenerative condition) treated with the substantially homogenouspopulation cells of the invention which co-express CD49c and CD90 orco-express CD49c, CD90 and telomerase.

The substantially homogenous population of cells which co-express CD49cand CD90 or which co-express CD49c, CD90 and telomerase whenadministered to the human may respond to cellular signaling andphysiological cues in the human and migrate to the area of injury ortrauma and, therefore, be used as delivery vehicles for proteins andgenes of interest.

In another embodiment, the invention is a method of treating a humansuffering from a neurological condition (e.g., spinal cord injury, anamyotrophic lateral sclerosis, a Parkinson's Disease, a stroke, atraumatic brain injury, a Fabry Disease condition, metachromaticdistropy, adrenal leukodystrophy, Canavan disease, Pelizaeus Merzbacher,Nieman-pick, a brain tumor) by culturing a source of a cell populationat a seeding density of less than about 100 cells/cm² under a low oxygencondition; selecting from the cultured source of the cell population, apopulation of cells which co-express CD49c and CD90; and administeringthe population of cells which co-express CD49c and CD90 to the human.

The transplantation of the substantially homogenous cell population ofthe invention into a patient suffering from a neurological condition mayresult in the differentiation of the cells of the invention into cellswhich normally function in the nervous tissue affected in the human withthe neurological condition thereby treating a myriad of neurologicalconditions including, for example, Parkinson's disease, ALS, spinal cordinjury, brain tumors, stroke. Similarly, the homogenous cell populationof the invention can be used to treat a human suffering from anon-neurological condition such as a burn, heart disease, diabetes,osteoarthritis and rheumatoid arthritis.

The cell populations of the invention may have the capacity to respondto intrinsic signals (e.g., at the sites of transplantation or whenincorporated into tissues and organs) and exogenous cues todifferentiate into numerous cell types (e.g., neuronal, glial,astrocytes, oligodendrocystes) in the human. The cell populations of theinvention can provide a readily available source of cells for use intreating humans. The cell populations of the invention can be readilyisolated from adult or embryonic tissues, proliferate at high rates,have large expansion potential, can be stable for long periods of time,can be responsive to exogenous signals and can produce sufficienttherapeutic quantities of molecules of interest.

Accordingly, another embodiment of the invention is a method of making acommitted progenitor cell by culturing (e.g., under a low oxygencondition, 5% oxygen) a source of a cell population (e.g., bone marrowcells, human bone marrow cells, fat, cord blood, skin) and selectingfrom the cultured source of the cell population cells which co-expressCD49c and CD90. The population of cells which co-express CD49c and CD90are modified to become committed progenitor cells. The selection ofcells from the cultured source of the cell population cells whichco-express CD49c and CD90 is achieved by a low oxygen condition (e.g.,oxygen below atmospheric oxygen, 5% oxygen).

“Committed progenitor cell,” as used herein, refers to a precursor cellobtained from a source (e.g., human bone marrow, fat, cord blood, skin)which develops into a cell for a particular purpose. A committedprogenitor cell can be, for example, a CD49c/CD90 cellderived from humanbone marrow which can differentiate or develop into, for example, aneuron, glial, astrocyte or oligodendrocyte cell.

In another embodiment, the invention is a method of treating a humansuffering from a neurological condition by culturing a source of a cellpopulation (e.g., bone marrow aspirates) and selecting (e.g., by a lowoxygen culture condition) from the cultured source of the cellpopulation, cells which co-express CD49c and CD90; co-express CD49c,CD90 and telomerase; or co-express CD49c, CD90 and a bone lineagemarker. The selected cells which co-express, for example, CD49c and CD90are modified to become a committed progenitor cell and administered to ahuman with a neurological condition (e.g., a spinal cord injury, anamyotrophic lateral sclerosis, a Parkinson's Disease, a stroke, atraumatic brain injury, a Fabry Disease condition, metachromaticdistropy, adrenal leukodystrophy, Canavan disease, Pelizaeus Merzbacher,Nieman-pick and a brain tumor).

Techniques to assess whether a cell of the substantially homogenouspopulation of cells of the invention which co-express CD49c and CD90;co-express CD49c, CD90 and telomerase; or CD49c, CD90 but does notexpress bone salioprotein (BSP) become committed progenitor cells arewithin the expertise of one of skill in the art. For example, cellswhich co-express CD49c and CD90 can be cultured, selected and modifiedto produce and express the neuronal cell markers, such as noggin,musashi or Sox2, which would indicate that the cells are committedneuronal progenitors cells. Techniques to determine whether a cell hasbecome a committed progenitor cell are well-established and known to oneof skill in the art (e.g., quantitative PCR, flow cytometry).

Cells which co-express CD49c and CD90 can be selected from a source of acell population for making the committed progenitor cells. Selectedcells which co-express CD49c and CD90 (also referred to herein as“selected cells”) can be, for example, modified to become committedprogenitor cells by culturing the selected cells in:

-   -   1. DMEM/F12/ITS/2 mM Glutamine/BSA1 mg/ml;    -   2. DMEM/F12/ITS/2 mM Glutamine/BSA1 mg/ml/0.25 ng/ml IL-β;    -   3. DMEM/F12/ITS/2 mM Glutamine/BSA1 mg/ml/2 ng/ml TNFα;    -   4. DMEM/F12/ITS/2 mM Glutamine/BSA1 mg/ml/100 ng/ml NT-3;    -   5. DMEM/F12/ITS/2 mM Glutamine/BSA1 mg/ml/100 ng/ml Noggin;    -   6. DMEM/F12/ITS/2 mM Glutamine/BSA1 mg/ml/100 ng/ml GDNF;    -   7. DMEM/F12/ITS/2 mM Glutamine/BSA1 mg/ml/20 ng/ml/bFGF;    -   8. DMEM/F12/ITS/2 mM Glutamine/BSA1 mg/ml/10 μM Forskolin;    -   9. DMEM/F12/ITS/2 mM Glutamine/BSA1 mg/ml/1 μM Bay K 8644;        and/or    -   10. DMEM/F12/B27/2 mM Glutamine/BSA1 mg/ml.    -   11. MEM-Alpha/4 mM Glutamine/10% ser lot selected fetal bovine        serum and 5 mM nifedipine

Selected cells can be used directly from cultures or stored for futureuse (e.g., by freezing in liquid nitrogen).

In one embodiment, the committed progenitor cells of the invention donot express CD34 and/or CD45. In another embodiment, the committedprogenitor cells of the invention express at least one trophic factorselected from the group consisting of BDNF, NGF, NT-3, IL-6, IL-7,IL-11, SCF, MCP-1, matrix metalloproteinase-9 (MMP-9) and Cystatin-C(e.g., BDNF, IL-6 and MCP-1).

In an additional embodiment, the invention is a method of treating ahuman suffering from a neurological condition, comprising culturing asource of a cell population (e.g., bone marrow, human bone marrow, fat,cord blood, skin) and selecting from the cultured source of the cellpopulation cells which co-express CD49c and CD90. The selected cells aremodified to become a committed progenitor cell. The committed progenitorcells are administered to a human.

In still another embodiment, the invention includes a pharmaceuticalcomposition comprising a substantially homogeneous cell population whichco-express CD49c and CD90 (e.g., between about 5×10⁵ to 2×10⁶ cells). Inone embodiment, the pharmaceutical composition has at least about 10⁵substantially homogeneous cells which co-express CD49c and CD90. Inanother embodiment, the pharmaceutical composition has at least about10⁶ substantially homogeneous cells which co-express CD49c and CD90. Thecells comprising the pharmaceutical composition can also not expressCD34 and/or CD45 and/or can express at least one trophic factor selectedfrom the group consisting of BDNF, NGF, NT-3, IL-6, IL-7, IL-11, SCF,MCP-1, matrix metalloproteinase-9 (MMP-9) and Cystatin-C.

In a further embodiment, the invention includes a pharmaceuticalcomposition comprising a substantially homogeneous cell population whichco-express CD49c, CD90 and telomerase.

The substantially homogenous cells of the invention which co-expressCD49c and CD90; co-express CD49c, CD90 and telomerase; or co-expressCD49c, CD90 but does not express BSP, can be administered to a humansuffering from a neurological condition. A “therapeutically beneficialamount” of the substantially homogenous population of cells of theinvention is a quantity sufficient to enhance neuronal function in asubject having a neurological condition (e.g., spinal cord injury) to beclinically relevant.

The cells of the invention can be, for example, transplanted or placedin the central (e.g., brain or spinal cord) or peripheral nervoussystem. The site of placement in the nervous system for the cells of theinvention is determined based on the particular neurological condition(e.g., direct injection into the lesioned spinal cord parenchyma,intra-thecal injection, or intravenous injection). For example, cells ofthe invention can be placed in or near the substantia nigra of patientssuffering from Parkinson's disease. Similarly, cells of the inventioncan be placed in or near the spinal cord (e.g., cervical, thoracic,lumbar or sacral) of patients suffering from a spinal cord injury.

The cells of the invention can be placed or transplanted in cavities orspaces of the central or peripheral nervous system. For example, thecells of the invention can be placed in the ventricles of the brain,subarachoid space of the spinal cord, or vertebral canal of the spinalcord. One skilled in the art would be able to determine the manner(e.g., needle injection or placement, more invasive surgery) mostsuitable for placement of the cells depending upon the location of theneurological condition and the medical condition of the patient.

In addition, routes of administration of the cells of the invention, orwhen cells of the invention are admixed with pharmaceutical carriers,encompassed by the present invention include, for example,intramuscular, intravenous, intraarterial, intraperitoneal, subcutaneousroutes or nasal administration.

The substantially homogenous cells of the invention which co-expressCD49c and CD90 can be administered alone or as admixtures withconventional excipients, for example, pharmaceutically, orphysiologically, acceptable organic, or inorganic carrier substancessuitable for enteral or parenteral application which do notdeleteriously react with the cells of the invention. Suitablepharmaceutically acceptable carriers include water, salt solutions (suchas Ringer's solution), alcohols, oils, gelatins and carbohydrates suchas lactose, amylose or starch, fatty acid esters, hydroxymethycellulose,and polyvinyl pyrolidine. Such preparations can be sterilized and, ifdesired, mixed with auxiliary agents such as lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, coloring, and/or aromatic substances and the likewhich do not deleteriously react with the cells of the invention.

When parenteral application is needed or desired, particularly suitableadmixtures for the cells are injectable, sterile solutions, preferablyoily or aqueous solutions, as well as suspensions, emulsions, orimplants, including suppositories and soaking in GELFOAM®. Inparticular, carriers for parenteral administration include aqueoussolutions of dextrose, saline, pure water, ethanol, glycerol, propyleneglycol, peanut oil, sesame oil and polyoxyethylene-block polymers.Pharmaceutical admixtures suitable for use in the present invention arewell-known to those of skill in the art and are described, for example,in Pharmaceutical Sciences (17th Ed., Mack Pub. Co., Easton, Pa.) and WO96/05309 the teachings of both of which are hereby incorporated byreference.

The substantially homogenous population of cells which co-express CD49cand CD90 can be used alone or in any combination when administered to ahuman suffering from a neurological condition. For example, steroids orpharmaceutical synthetic drugs can be co-adminstration with the cells ofthe invention. Likewise, treatment of spinal cord injury can include theadministration/transplantation of the cells of the invention in a humanwhose spine has been physically stabilized.

The dosage and frequency (single or multiple doses) of theadministration or transplantation of the cells to a human, including theactual number of cells transplanted into the human, can vary dependingupon a variety of factors, including the particular condition beingtreated (e.g., neurological condition, cardiac condition, degenerativecondition, acute injury) size, age, sex, health, body weight, body massindex, diet of the human, nature and extent of symptoms of theneurological condition being treated (e.g., early onset Parkinson'sdisease versus advanced Parkinson's disease; spinal cord trauma versuspartial or complete severing of the spinal cord), kind of concurrenttreatment (e.g., steroids), complications from the neurologicalcondition, extent of tolerance to the treatment or other health-relatedproblems.

Humans with a neurological condition can be treated for days (e.g., 30)with cells of the invention (e.g., about 10⁶ cells), by a several routesof administration (e.g., intrathecal, intravenous).

It is also envisioned that the methods of the invention can be employedto treat neurological conditions in mammals other than human mammals.For example, a non-human mammal in need of veterinary treatment, e.g.,companion animals (e.g., dogs, cats), farm animals (e.g., cows, sheep,pigs, horses) and laboratory animals (e.g., rats, mice, guinea pigs).

The present invention is further illustrated by the following examples,which are not intended to be limiting in any way.

EXEMPLIFICATION EXAMPLE 1 Isolation of a Adherent as Colony FormingUnits of Cells or “CFUs” from Bone Marrow Aspirates Following Red BloodCell Lysis

Bone marrow cells were aspirated from the iliac crest of healthy adulthuman volunteers. The red blood cell component of the aspirate was lysedby mixing the aspirate with an ammonium chloride buffer consisting of155 mM ammonium chloride, 10 mM potassium bicarbonate and 0.1 mM EDTA(ethylenediaminetetraacetic acid), pH 7.2, at a 1:20 ratio of marrowaspirate to buffer. The resulting cell suspension was vortexed for 2seconds, incubated for 2 minutes at ambient temperature and thencentrifuged (10 minute at 500×g). The resulting mononuclear cell pelletwas resuspended in complete medium and centrifuged (10 minutes at500×g). Complete media is Minimal Essential Medium-alpha (Gibco BRL,Rockville, Md.) supplemented with 4 mM glutamine and 10% sera-lotselected fetal bovine serum (FB S, Gibco BRL, Rockville, Md.). The cellpellet was then re-suspended in the complete medium and centrifuged asecond time (10 minutes at 500×g).

The resulting pellet was re-suspended in the complete medium and thenumber of viable cells was determined by trypan blue-exclusion. Themononuclear cell suspension was then seeded in tissue culture-treatedT75 flask at a density of 50,000 cells/cm2 and incubated at 37° C. in anatmosphere consisting of 5% carbon dioxide, 5% oxygen, and 90%nitrogen/air. On the fifth day of culture, the non-adherent cells andconditioned media (also referred to herein as “spent media”) wereaspirated from the flasks and the adherent cells re-fed with freshcomplete medium. The adherent colony forming units (CFUs) were expandedfor an additional 3-5 days.

The generation of CFUs was monitored in 6-well plates concurrentlyinitiated under identical conditions to the T75 flasks. The spent mediumwas removed from the 6-well plates and the adherent cells were fixed for5 minutes in 100% methanol, and then stained with methylene blue tovisualize the CFUs. An initial seeding density of 75,000 cells/cm²efficiently generated CFUs. After processing the bone marrow aspirate byeither FICOLL® density gradient separation or ammonium chloride lysis,CFU efficiency was dramatically affected by oxygen concentration.

After 7 days in culture, the purity (percentage of cells whichco-express CD49c and CD90) of the CFUs generated was determined by flowcytometry. T75 flasks were washed twice with Hank's Balanced SaltSolution (HBSS; CellGro Technologies) and treated with 0.1% Trypsin/1 mMEDTA solution (Life Technologies) for 10 minutes at 37° C. Cultures wereremoved from incubator and 10 mL of complete medium was added. Cellswere triturated from the flask, transferred to a 50 mL centrifuge tubeand centrifuged (500×g for 5 minutes). The resulting pellet wasresuspended in 10 mL of HBSS.

Resuspended cells (approximately 10⁶) were aliquoted into 12×75 mm FlowCytometry tubes and repelleted at 500×g for 5 minutes. The HBSS wasremoved and 25 mL of the following antibodies (all obtained from BectonDickenson), alone or in combination, were placed into each tube: mouseIgG1k-FITC or -PE (clone MOPC-21) CD49c-PE (cl. C3II.1), CD90-FITC (cl.5E10), CD45-FITC or -PE (cl. HI30). Tubes were gently vortexed andincubated for 30 minutes at 4° C. Cells were then washed in HBSS/1%bovine serum albumin, centrifuged (30 min, 4° C.) and the resultingcellular pellet fixed by the addition of 250 microliters of 2%paraformaldhyde/HBSS. Flow cytometric analysis was performed employing aBecton-Dickenson FACSVantage SE cytometer and analyzed using CELLQUEST®software. FIG. 1 depicts results representing data collected from2,500-10,000 events per panel. After compensation for non-specificantibody staining using mouse IgG1 isotype controls, cellular expressionof CD45, CD49c and CD90 in the cultured bone marrow cells was assessed.The adherent population derived from mononuclear cells initiallypurified using ammonium chloride lysis contained approximately 70% CD49cpositive cells at a similar stage of culture (FIG. 1A). The majority ofcells that did not express CD49c were positive for expression ofhematopoetic/myeloid lineage marker CD45 (FIG. 1A, LR quadrant),demonstrating that the CD49c positive cell population derived from humanbone marrow isolated was not directly related to known hematopoieticprecursors. More than 94% of the adherent population was CD90 and CD49cpositive (FIG. 1B).

EXAMPLE 2 Isolation of a Adherent CFUs from Bone Marrow AspiratesFollowing Density Separation

Bone marrow cells were aspirated from the iliac crest of healthy adulthuman volunteers. The bone marrow aspirate was diluted with calcium andmagnesium free phosphate buffered saline (PBS) to achieve a mononuclearcell concentration of 7×10⁶ cells/mL and overlaid onto an equal volumeof Histopaque® 1.119 (Sigma, St. Louis, Mo.) and centrifuged (30 min at700×g). The resulting mononuclear cell fraction was transferred to aclean centrifuge tube containing PBS and centrifuged (10 minutes at500×g). The cell pellet was re-suspended in PBS and centrifuged (10minutes at 500×g). The supernatant was aspirated from the cell pelletand the cells re-suspended in complete media.

The number of viable cells in the resulting cell suspension wasdetermined by trypan blue-exclusion. The cell suspension was then seededin tissue culture-treated T75 flasks at a density of 50,000 cells/cm2and incubated at 37° C. in an atmosphere of 5% carbon dioxide, 5% oxygenand 90% nitrogen/air. On the fifth day of culture, the non-adherentcells and conditioned media (also referred to herein as “spent media”)was aspirated from the flasks and the adherent cells re-fed with freshcomplete medium. The adherent CFUs were expanded for an additional 3-5days.

Cytometry analysis of the CFU generated showed that approximately 50% ofthe adherent population expressed the marker CD49c at 7 days in vitro(FIG. 2A, sum of UL and UR quadrants). The majority of cells that didnot express CD49c were positive for expression of hematopoetic/myeloidlineage marker CD45 (FIG. 2A, LR quadrant), demonstrating that the CD49cpositive cell population derived from human bone marrow isolated by thisprocedure was not directly related to known hematopoietic precursors.More than 91% of the adherent population was CD90 and CD49c positive(FIG. 2B).

EXAMPLE 3 Production of Primary and Master Cell Banks from CFUs

After 7-10 days in culture, the CFUs generated using the methodsdescribed in Example 1 were removed from the T75 flasks with a 0.25%trypsin/1 mM EDTA solution (Life Technologies). After 10 minutes at 37°C., the trypsin was inactivated with 10 mL of complete medium. The cellswere washed once with HBSS and re-suspended in Glycerol Cell FreezingMedium® (Sigma Chemical Co.). Aliquots (referred to herein as “thePrimary Cell Bank”) of the suspension consisting of 4.0×10⁵ cells/vialwere cooled with liquid nitrogen vapor at 1° C./minute using a CryoMed(Forma) controlled rate freezer and the stored in a Cryo Plus liquidnitrogen storage tank (Forma).

An aliquot of cells was removed from the Primary Cell Bank and culturedat a density of 30 cells/cm² in 500 cm² tissue culture-treated plates(Coming) in complete medium and incubated at 37° C. in an atmosphereconsisting of 5% carbon dioxide, 5% oxygen, and 90% nitrogen/air. Aftertwo weeks of culture, cells were removed from the plates with trypsinand were cryopreserved at 4.0×10⁵ cells/vial (referred to herein as “theMaster Cell Bank”).

The purity of the cells (percentage of cells which co-expressCD49c/CD90) in the Master Cell Bank was determined by flow cytometryusing the same method as above. The vast majority (>98%) of theresulting population expressed CD90 (FIG. 1C) and virtually lacked anyexpression of the myeloid-related marker CD45 (FIG. 1C, LR quadrant).Thus, the expansion procedure as described herein produces asubstantially homogenous population of adherent cells which co-expressCD49c and CD90 and lack significant expression of the marker CD45.

Similarly, the master cell bank generated from the CFU derived usingmethod of Example 2 showed that the majority of cells (>98.8%) of theresulting cell population expressed CD90 (FIG. 2C) and virtually lackedany expression of the myeloid-related marker CD45 (FIG. 2C, LRquadrant). Thus, the expansion procedure as described herein generates asubstantially homogenous population of adherent cells which co-expressCD49c and CD90 and lack significant expression of the marker CD45.

EXAMPLE 4 Expansion Capability of the Cell Population which Co-ExpressCD49c and CD90

A Primary Cell Bank of CFUs was derived from 25 mLs of bone marrowaspirate and stored as frozen aliquots using the methods of Examples 1and 2. An aliquot was thawed, expanded and frozen to generate the MasterCell Bank as described in Example 3. An aliquot of cells was removedfrom the Master Cell Bank and cultured at a density of 30 cells/cm² in500 cm² tissue culture-treated plates (Coming) in complete medium andincubated at 37° C. in an atmosphere consisting of 5% carbon dioxide, 5%oxygen, and 90% nitrogen/air. After two weeks of culture, cells wereremoved from the plates with trypsin and cryopreserved at 2-10×10⁶cells/vial. The process was repeated in succession to produce additionalCell Banks of cells which co-express CD49c and CD90.

The cell number generated from a single aliquot at the end of eachsuccessive expansion was determined by trypan exclusion and multipliedby the number of aliquots to calculate the yield (FIG. 3). Foursuccessive expansions can potentially generate up to 1×10¹⁷ cells from25 mLs of bone marrow aspirate obtained from a single donor. The numberof cell doublings was calculated from the cell yields using thefollowing formula: (Log(end cell #)−Log(starting cell #)/Log(2) (“(2)”denotes doubling). The cell population underwent about 8 doublingsduring each expansion (FIG. 4). The doubling rate (# days inculture×24/doublings) was 30 hrs and remained constant for at least 50doublings. Even after 30 doublings, the population uniformly retainedthe characteristic morphology of small, dividing cells without apparentevidence of the enlarged, flat morphology of aged orterminally-differentiated cells.

EXAMPLE 5 Expression of Transcripts Encoding Regulators of Cell Growthand Osteoblast Differentiation by Cell Populations which Co-ExpressCD49c and CD90

The expression of transcripts for telomerase, p21, p53, CBFA1 and BSPwere determined using quantitative polymerase chain reaction (qPCR).Briefly, Master Cell Bank of CFUs were derived from a bone marrowaspirate and stored as frozen aliquots using the method of Example 1. Analiquot was thawed, cultured at a density of 30 cells/cm² in tissueculture-treated T75 flasks in complete medium and incubated at 37° C. inan atmosphere consisting of 5% carbon dioxide, 5% oxygen, and 90%nitrogen/air.

After two weeks of culture, the cells were seeded in 96 well plates at3000 cells/well. RNA was isolated using the QIAGEN RNeasy reagents andthe Qiagen Biorobot 3000. An aliquot of the eluted RNA was used tosynthesize cDNA. RNA was mixed with Promega MMLV, dNTPS, decamers andRNasin and incubated at 37° C. for 1 hour, followed by heatinactivation. For quantitative PCR, cDNA samples were combined withApplied Biosystems SYBR Green PCR Core reagents and amplicon specificprimers, as described below, in a 384 well format. The 384 well platewas then transferred to the Applied Biosystems ABI Prism 7900 for qPCRanalysis. The qPCR program entailed a 2 minute cycle at 50 degrees,followed by a 10 minute cycle at 95 degrees to activate the polymerase.This was than followed by 40 amplification cycles consisting of 15seconds of melting at 95 degrees and one minute of extension/annealingat 60 degrees.

Cycle threshold values were converted into relative transcript numberusing a standard curve then normalized using the corresponding 18s. Dataare expressed as a ratio of transcript per 10⁶ 18s transcripts. Thename, Genebank ID, bp location and sequence of the qPCR primers are asfollows: 18s-1F, K03432, 1742-1760 bp, (SEQ ID NO:1) 5′-ATG GGG ATC GGGGAT TGC A-3′; 18s-1R, K03432, 1871-1890 bp, (SEQ ID NO:2) 5′-CCG ATC CGAGGG CCT CAC TA-3′; BSP-1F, NM000582, 483-508 bp, (SEQ ID NO:3) 5′-CACTCC AGT TGT CCC CAC AGT AGA CA3′; BSP-1R, 611-632 bp, (SEQ ID NO:4)5′-TCG CTT TCC ATG TGT GAG GTG A-3′; CBFA1-1F, L40992, 389-407 bp, (SEQID NO:5) 5′-GGC CGG AGT GGA CGA GGC AA-3′; CBFA1-1R, L40992, 504-529 bp,(SEQ ID NO:6) 5′-CAT CAA GCT TCT GTC TGT GCC TTC TG-3′; p21-1F, S67388,52-72 bp, (SEQ ID NO:7) 5′-ACC GAG GCA CTC AGA GGA GGC-3′; p21-1R,S67388, 171-191 bp, (SEQ ID NO:8) 5′-GCC ATT AGC GCA TCA CAG TCG-3′;p53-qFP4, M14694, 521-545 bp, (SEQ ID NO:9) 5′-GAT GTT TTG CCA ACT GGCCAA GAC C-3′; p53-qRP4, M14694, 674-698 bp, (SEQ ID NO:10) 5′-AGG AGGGGC CAG ACC ATC GCT ATC T-3′; Telo-1F; AF015950, 1500-1525 bp, (SEQ IDNO:11) 5′-ACA ACG AAC GCC GCT TCC TCA GGA AC-3′; and Telo-1R, AF015950,1625-1650 bp, (SEQ ID NO:12) 5′-GCC GGA ACA CAG CCA ACC CCT GG-3′.

Telomerase activity is necessary for maintaining telomeres, which areDNA sequences located at the ends of chromosomes. Since most human cellslack telomerase, the telomeres shorten with each division until thecells growth arrest (Harley, C. B., Mutation Research 256(2-6):271-282(1991); Hara, E. et al., Biochem Biophys Res Commun 179(1):528-534(1991); Shay, J. W. et al., Exp Cell Res 196(1):33-39 (1991)). Cellpopulations which co-express CD49c and CD90 express telomerase at thelevel of approximately 13 transcripts/10⁶ transcripts of 18S rRNA, whichis consistent with the finding that this cell population continued toproliferate at a constant rate.

The p53 tumor suppressor plays a key role in the cell's response to DNAdamage and inactivation of this gene is an important step incarcinogenesis. p53 expression is upregulated in response to DNA damage.Its ability to prevent the proliferation of defective cells involves theactivation of several growth arrest genes including p21 (Burns, T. F. etal., Oncogene 20(34):4601-4612 (2001)). The substantially homogenouscell population of the invention which co-express CD49c and CD90expressed about 670 p53 transcripts/10⁶ transcripts of 18S rRNA. Thislevel of p53 shows that the tumor suppressor p 53 was not induced.

p21 is a potent cell cycle inhibitor and its expression during the cellcycle is tightly regulated at the transcriptional level (Gartel, A. L.et al., Exp Cell Res 246(2):280-289 (1999)). p21 is induced in growtharrested cells in response to oxidative stress in addition to DNA damage(Yin, Y., et al., Mol Carcinog 24(1): 15-24 (1999)). The substantiallyhomogenous cell population of the invention which co-express CD49c andCD90 expressed p21 about 1690 transcripts/10⁶ transcripts of 18s rRNA.This level of p21 shows that p21 was not induced and is consistent withboth a low level of p53 and the short doubling time measured in Example4.

The transcription factor, CBFA1, is necessary for osteoblastdifferentiation and bone formation (Otto, F., Cell 89(5):765-771(1997)). Bone sialoprotein (BSP) is a prominent, mineral-associatedprotein in the extracellular matrix of bone and is expressed by fullydifferentiated osteoblasts (Benson, M. D., et al., J Biol Chem275(18):13907-13917 (2000)). The substantially homogenous cellpopulation of the invention which co-express CD49c and CD90 expressedabout 130 CBFA1 transcripts/10⁶ transcripts of 18S rRNA and BSP wasabsent, which show that the cell population of the invention representsa progenitor that has not significantly differentiated into osteoblasts.Osteoblast differentiation is described in Ducy, P., Dev Dyn219(4):461-471 (2000)).

EXAMPLE 6 Secretion of Neurotrophic Factors and Cytokines by aSubstantially Homogenous Cell Population which Co-Express CD49c and CD90

An aliquot of the Master Cell Bank generated in Example 1 was thawed andplated onto T75 flasks at 2500 cells/cm² with complete medium andincubated at 5% O₂. The following day the medium was removed andreplaced with fresh complete medium. The supernatant was collected 8hours later from T75 and the cells were counted (Cell count=280,000cells). The supernatant was aliquoted to 1 ml tubes and stored at −20°C. Another T75 was processed the same way 3 days later (Cell count=2.43million). Supernatants were later thawed at room temperature and assayedby ELISA for secretion of the following neurotrophicfactors/cytokinesusing commercially available kits: BDNF (Chemicon), NGF (Chemicon),MCP-1 (R and D Systems), and IL-6 (R and D Systems). Multiple dilutionswere performed on supernatant to ensure that measured values fell withinstandard ranges of the assay. In addition, media obtained from controlcells secreting previously determined amounts of cytokine were run inparallel to assure assay validity. Values were obtained by normalizingraw data derived from ELISA to standard time (24 hours) and cell number(1 million) and are thus expressed as “picograms of cytokine secretedper 1 million cells per 24 hour period as follows: Cytokine AmountSecreted (pg/10⁶ cells/day MCP-1 1009.15 IL-6 18567.60 BDNF 8.88 NGF80.12

EXAMPLE 7 Transplantation of a Substantially Homogenous Cell Populationof the Invention which Co-Express CD49c and CD90 into an Acute RatSpinal Cord Injury Model

Following traumatic injury to the spinal cord neuronal death,inflammation and progressive loss of damaged neurons ensue overtime. Asubstantially homogenous cell population of the invention whichco-express CD49c and CD90 improved outcome during acute neurologicinjury. A cell population prepared as described in Example 1 wastransplanted into contused spinal cord of adult female Sprague-Dawleyrats.

The thoracic spinal cord of Sprague-Dawley rats was exposed bylaminectomy at the level of T10 under general anesthesia. After thelaminectomy is completed, a 10 gm rod was dropped from a height of 25 mmto produce a spinal cord injury of moderate severity onto the exposedspinal cord using the NYU spinal cord impactor (Constantini, S. et al.,J Neurosurg 80(1):97-111 (1994)). During surgery, the body temperatureof the rats was kept at 37° C. During recovery, rats were placedovernight in a temperature and humidity controlled chamber. Seven daysafter impact injury, the spinal cords were re-exposed. Using a 50 mL gastight Hamilton syringe (VWR Scientific Products, Bridgeport, N.J.) witha 30 gauge needle, 250,000 cells of the invention at a concentration of25,000/μl were transplanted into the spinal cord. The cells wereinjected into the epicenter of the syrninx at the T10 level at a rate of2 mL/minute. The needle was left in place for additional 5 minutesbefore it was removed from the spinal cord. Following surgery, allanimals received methylprednisolone (30 mg/kg, i.v.) immediatelyfollowing surgery. To prevent immun-rejection, Cyclosporin A (CsA) wasgiven subcutaneously at 10 mg/kg 3 days prior to the day oftransplantation and maintained thereafter.

The Basso-Beattie-Bresnahan openfield locomotor test (BBB Test) (Basso,D. B. et al., J Neurotrauma 12(1):1-21 (1995)) were performed the daybefore transplantation (day 6 after injury). Behavioral testing wasperformed for each hindlimb weekly using the BBB scores. Scoring wasperformed blinded to the treatment status. At 19 days after contusion,animals that had received a substantially homogenous population of cellswhich co-express CD49c and CD90, showed greater improvement on the BBBscore than animals received only PBS (10.6±1.2 vs 8.5±0.3).

At 2 weeks after transplantation, contused spinal cords from someanimals were removed and examined for evidence on nerve fiberregeneration. SMI 32 (Stemberger Monoclonals, Inc, Lutherville, Md.)antibody was used to immunostain for regenerating fibers in the syrinxat a dilution of 1:4000. Numerous fibers were observed growing into thecontused syrinx.

EXAMPLE 8 Expression of Transcripts Encoding Regulators of NeuronalDifferentiation by a Substantially Homogenous Population of Cells whichCo-Express CD49c and CD90

The expression of transcripts for Sox-2 and Musashi were determinedusing quantitative polymerase chain reaction (qPCR). Briefly, MasterCell Bank of CFUs were derived from a bone marrow aspirate and stored asfrozen aliquots using the method of Example 1. An aliquot was thawed andthe cells were seeded in 96 well plates at 2000 cells/well in completemedia and incubated at 37° C. in an atmosphere consisting of 5% carbondioxide, 5% oxygen and 90% nitrogen/air for 3 days. On the third day ofculture, cells were treated with 5 μM nifedipine (an L-type calciumchannel blocker). After 24 hours of treatment, RNA was isolated usingthe QIAGEN®RNeasy reagents and the Qiagen Biorobot 3000. An aliquot ofthe eluted RNA was used to synthesize cDNA. Specifically, RNA was mixedwith Promega MMLV, dNTPS, decamers and RNasin and incubated at 37° C.for 1 hour, followed by heat inactivation.

For quantitative PCR, cDNA samples were combined with Applied BiosystemsSYBR Green PCR Core reagents and amplicon specific primers, as describedbelow, in a 384 well format. The 384 well plate was then transferred tothe Applied Biosystems ABI Prism 7900 for qPCR analysis. The qCPRprogram entailed a 2 minute cycle at 50 degrees, followed by a 10 minutecycle at 95 degrees to activate the polymerase. This was then followedby 40 amplification cycles consisting of 15 seconds of melting at 95degrees and one minute of extension/annealing at 60 degrees. Cyclethreshold values were converted into relative transcript number using astandard curve then normalized using the corresponding 18s. Data areexpressed as a ratio of transcript per 10⁶ 18s transcripts.

The name, Genebank ID, bp location and sequence of the qPCR primers areas follows: 18s-1F, K03432, 1742-1760 bp, (SEQ ID NO:1) 5′-ATG GGG ATCGGG GAT TGC A-3′; 18s-1R, K03432, 1871-1890 bp, (SEQ ID NO:2) 5′-CCG ATCCGA GGG CCT CAC TA-3′; Sox-2F, Z31560, 517-541 bp, (SEQ ID NO:13) 5′-GGCAGC TAC AGC ATG ATG CAG GAC C-3′; Sox-2R, 624-647 bp, (SEQ ID NO:14)5′-CTG GTC ATG GAG TTG TAC TGC AGG-3′; Musashi-1F, AB012851, 370-389 bp,(SEQ ID NO:15) 5′-CAA GAT GGT GAC TCG AAC GA-3′; Musashi-1R, 480-499 bp,(SEQ ID NO:16) 5′-GGT TTT GTC AAA CAT CAG CA-3′.

The gene Sox-2 encodes a conserved nuclear transcription factor relatedto the Mammalian Testis Determining Gene that is expressed throughoutthe neural tube during brain development and is essential for thesurvival of primitive neural ectoderm (Uwanogho, Mech. Dev. 49:23-36(1995)). In addition, expression of Sox-2 persists beyond development inrestricted populations of adult neural stem cells, suggesting a furtherrole for this factor in regulating neural fate (Zappone, Development127:2367-2382 (2000)). Under unstimulated conditions in complete media,the cells which co-express CD49c and CD90 expressed approximately 4Sox-2 transcripts/10⁶ transcripts of 18S RNA. However, in response tonifedipine, the cells which co-express CD49c and CD90 expressed 28 Sox-2transcripts/10⁶ transcripts of 18S RNA, an approximate 7-fold increase.This increase in Sox-2 expression suggests that, in response to certainepigenetic treatments, the cells which co-express CD49c and CD90 maydisplay traits associated with early neural populations.

The gene Musashi encodes an RNA-binding protein that is highly expressedwithin the developing nervous system and, like Sox-2, is also expressedin mammalian neural stem cells (Sakakibara, Dev Biol 176:230-242(1996)). Furthermore, expression of Musashi is required for normaldevelopment of multiple neuronal population (Nakamura, Neuron 13:67-81(1994)). Under unstimulated conditions in complete media, the cellswhich co-express CD49c and CD90 expressed approximately 0.005 Musashitranscripts/10⁶ transcripts of 18S RNA. However, in response to 24 hoursof stimulation with nifedipine, the cells which co-express CD49c andCD90 expressed 0.093 transcripts/10⁶ transcripts of 18S RNA, anapproximate 17-fold increase. This increase in Sox-2 expression suggeststhat, in response to certain epigenetic treatments, the cells whichco-express CD49c and CD90 may display traits associated with earlyneural populations.

Equivalents

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method of making a substantially homogenous cell population which co-express CD49c and CD90, comprising the steps of: a) culturing a source of the cell population at a seeding density of less than about 100 cells/cm² under a low oxygen condition; and b) selecting from the cultured source of the cell population, cells which co-express CD49c and CD90.
 2. The method of claim 1, wherein the source of the cell population is bone marrow.
 3. The method of claim 1, wherein the bone marrow is human bone marrow.
 4. The method of claim 1, wherein the low oxygen condition is less than about 15% oxygen.
 5. The method of claim 4, wherein the low oxygen condition is about is less than about 10% oxygen.
 6. The method of claim 1, wherein the low oxygen condition is about 5% oxygen.
 7. The method of claim 2, further including lysing the bone marrow prior to culturing the bone marrow.
 8. The method of claim 2, further including fractionating the bone marrow prior to culturing the bone marrow.
 9. The method of claim 8, wherein in the bone marrow is fractionated by passage through a density gradient.
 10. The method of claim 8, wherein the bone marrow is fractionated by NH₂Cl lysis.
 11. The method of claim 8, wherein the bone marrow is fractionated by fluorescent activated sorting.
 12. The method of claim 8, wherein the bone marrow is fractionated by magnetic sorting.
 13. The method of claim 1, wherein the cells which co-express CD49c and CD90 do not express CD34 and/or CD45.
 14. The method of claim 1, wherein the cells selected from the cultured source express at least one trophic factor selected from the group consisting of BDNF, IL-6, NGF and MCP-1.
 15. A method of making a substantially homogenous cell population which co-express CD49c and CD90, comprising the steps of: a) culturing a source of the cell population at a seeding density of less than about 100 cells/cm² under a low oxidative stress condition; and b) selecting from the cultured source of the cell population, cells which co-express CD49c and CD90.
 16. The method of claim 15, wherein the source of the cell population is bone marrow.
 17. The method of claim 15, wherein the bone marrow is human bone marrow.
 18. A method of making a substantially homogenous cell population which co-express CD49c and CD90, comprising the steps of: a) culturing a source of the cell population at a seeding density of less than about 50 cells/cm² under a low oxidative stress condition; and b) selecting from the cultured source of the cell population, cells which co-express CD49c and CD90.
 19. A method of making a substantially homogenous cell population which co-express CD49c and CD90, comprising the steps of: a) culturing a source of the cell population at a seeding density of less than about 30 cells/cm² under a low oxidative stress condition; and b) selecting from the cultured source of the cell population, cells which co-express CD49c and CD90.
 20. A method of making a substantially homogenous cell population which co-express CD49c and CD90, comprising the steps of: a) culturing a source of the cell population at a seeding density of less than about 50 cells/cm² under a low oxygen condition; and b) selecting from the cultured source of the cell population, cells which co-express CD49c and CD90.
 21. A method of making a substantially homogenous cell population which co-express CD49c and CD90, comprising the steps of: a) culturing a source of the cell population at a seeding density of less than about 30 cells/cm² under a low oxygen condition; and b) selecting from the cultured source of the cell population, cells which co-express CD49c and CD90. 